Antiviral compounds

ABSTRACT

The macrolide FK506, produced by Fujisawa Pharmaceuticals is effective in inhibiting the replication of hepatitis D virus. It is believed that replication is inhibited either by virtue of the ability of FK506 to inhibit proline isomerase or otherwise to interfere with the function of a C-terminal proline in a replication factor or by virtue of its interference with RNA replication directly.

This invention was made with government support under grant no. AI-22470awarded by the National Institutes of Health. The government has certainrights in this invention.

This application is a File Wrapper continuation of application Ser. No.08/144,759, filed Oct. 27, 1993, now abandoned.

TECHNICAL FIELD

The invention is directed to inhibitors of viral replication. Inparticular, it concerns the use of compounds related to theimmunosuppressant macrolide FK506 to interfere with replication.

BACKGROUND ART

The macrolide FK506, which is of the formula: ##STR1## is manufacturedby Fujisawa Pharmaceutical Company Limited, and is a knownimmunosuppressant. It is a member of class of immunophilins described bySchreiber, S. L. in Science (1991) 251:283-287. Other members of thisclass include rapamycin, cyclosporin A, and an additional macrolidedesignated 506BD. However, the present applicant is unaware of anysuggestion that FK506, which is also known to be a proline isomerase (orrotamase) inhibitor, or the other compounds described in this articlewould have antiviral activity. Unexpectedly, FK506 was found to becapable of inhibiting hepatitis Delta virus (HDV) replication.

U.S. Pat. No. 5,196,437 describes the ability of compounds related toFK506 to regenerate liver which has been negatively affected byhepatitis B and non A/non B. However, this appears to be unconnectedwith any direct effect on the virus.

DISCLOSURE OF THE INVENTION

The invention is directed to a method to inhibit viral replicationillustrated by the ability of FK506 to inhibit hepatitis D virusreplication. The surprising ability of FK506 to do this suggests thatreplication of a variety of viruses which share features with HDV may beinhibited by compounds related to FK506, provided at least one ofseveral specific conditions is met. FK506 is known to have prolineisomerase inhibiting activity. HDV antigen behaves as a transcriptionfactor (defined herein as a protein that plays a key role in nucleicacid polymerization) and contains a C-terminal proline essential forreplication, independent of the preceding amino acid sequence.Therefore, the ability of FK506 to inhibit HDV replication suggests thatcompounds which inhibit proline isomerases or which otherwise interferewith the function of a C-terminal proline in an essential replicationfactor will inhibit replication of viruses that replicate via at leastone such transcription factor.

Accordingly, in one aspect, the invention is directed to a method toinhibit viral replication which method comprises contacting a virus or acell in which a virus replicates, where the virus contains at least onetranscription factor which has a C-terminal proline, with an effectiveamount of at least one proline isomerase inhibitor such as FK506 or ofan inhibitor of proline recognition or function. In another aspect, theinvention is directed to a method to inhibit viral replication whereinthe virus, like HDV, replicates through an RNA intermediate, whichmethod comprises contacting the target virus or a cell in which thevirus replicates with FK506 or its acyl esters.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the effect of FK506 on HDV genereplication.

MODES OF CARRYING OUT THE INVENTION

In the illustration below, it is demonstrated that FK506, a prolineisomerase inhibitor, is capable of inhibiting HDV genome replication.This surprising result leads to the conclusion that viruses whichreplicate in a manner similar to HDV will be similarly affected bycompounds related to FK506. The features of HDV replication which aresignificant for interaction with FK506 include replication through anRNA intermediate and/or the presence of an essential transcriptionfactor having a C-terminal proline.

Viruses known to replicate through an RNA intermediate include thosebelonging to the picornavirus, togavirus, bunyavirus, orthomyxovirus,paramyxovirus, rhabdovirus, retrovirus, arenavirus, and hepatitis Bvirus families.

Viruses known to contain an essential transcription factor with aC-terminal proline include human adenovirus, feline leukemia virus,vaccinia virus, papilloma virus, hepatitis C virus, hepatitis B virus,human immunodeficiency virus, varicella zoster virus, and bunyavirus.

For any proposed target virus, the ability to ascertain these featuresis within the skill of the art and provides a basis for concluding ornot concluding that the relevant class of antiviral compounds will beeffective.

FK506 has the structure set forth in the Background section above. FK506contains three free hydroxyl groups; each hydroxyl group independentlymay be esterified with an acyl group of 1-6C. By "acyl (1-6C)" is meanta saturated or unsaturated moiety of the formula RCO wherein R issaturated or unsaturated hydrocarbyl. Typical such acyl groups includeacetyl, penten-1-oyl, propanoyl, 2-methyl propanoyl and the like.

Proline isomerase inhibitors other than FK506 include rapamycin,cyclosporin A, 506BD and their acyl derivatives. All of these compoundsare useful in the invention.

The following example is intended to illustrate but not to limit theinvention.

EXAMPLE 1

Inhibition of HDV with FK506

HDV gene replication was assayed by the method of Glenn, J. S., et al. JVirol (1991) 65:2357-2361. FK506 was supplied at various concentrationsinto the replication assays.

One day post delivery of S-genome RNA to GAG cells, the GAG cells weresplit equally into 60 mm dishes, which were maintained at specificconcentrations of FK506 ranging from 0.1 nM to 10 μM until harvest onday 7. The harvested RNA was assayed by Northern blot to detect HDVgenome replication. The Northern blots include a control probe for hostcell RNA (Signal Recognition Particle Receptor mRNA) to permitnormalizing total RNA levels. Phosphoimaging analysis permittedsensitive quantitation and comparison of levels of viral replication.

The results of three experiments are shown in FIG. 1; each bar reflectsthe level of HDV genome replication at the indicated drug concentration.The solid and striped columns represent results from duplicate dishesshowing the degree of variability. The stippled columns representresults obtained in a previous experiment conducted two monthspreviously when the drug was freshly constituted.

The results show that nM concentrations of FK506 achieve about 50%inhibition of HDV replication.

I claim:
 1. A method to inhibit the replication of a virus that requiresproline isomerase for replication, said method comprises the step ofcontacting a cell in which said virus replicates with an amount of aproline isomerase inhibitor sufficient to inhibit replication.
 2. Amethod to inhibit the replication of a virus that requires prolineisomerase for replication, said method comprises the step of contactinga cell in which said virus replicates with an amount of a compound ofthe formula: ##STR2## or a pharmaceutically acceptable acyl (1-6C) esterthereof sufficient to inhibit replication.
 3. A method to inhibit thereplication of a virus that requires proline isomerase for replication,said method comprises the step of contacting a cell in which said virusreplicates with an mount of a compound selected from the groupconsisting of FK506 rapamycin and 506BD sufficient to inhibitreplication.
 4. A method to inhibit the replication of hepatitis Dvirus, said method comprises the step of contacting a cell in which saidvirus replicates with an amount of a proline isomerase inhibitorsufficient to inhibit replication.
 5. A method to inhibit thereplication of hepatitis D virus, said method comprises the step ofcontacting a cell in which said virus replicates with an amount of acompound of the formula sufficient to inhibit replication: ##STR3## or apharmaceutically acceptable acyl (1-6C) ester thereof sufficient toinhibit replication.